Femtosecond cellular transfection using novel laser beam geometries

Abstract

In this thesis, femtosecond (fs) cellular transfection of Chinese Hamster Ovary (CHO) cells was performed using a tightly focused Gaussian beam. The beam focus was positioned on the cell membrane and three laser doses, each of 40 ms duration, were delivered allowing for the formation of a highly localized pore on the cell membrane. The membrane pore, induced as a result of a multiphoton process known as photoporation, permitted the surrounding DNA to diffuse into the cell cytoplasm. 48 hours after laser irradiation, the viable photoporated cells expressed a red fluorescent protein. The topography of a photoporated cell, targeted with tightly focused fs pulses, was also monitored as a function of the input power using Atomic Force Microscopy. Following this, I generated and implemented a “non-diffracting” quasi-Bessel beam (BB) by means of a conical shaped lens, the axicon, which successfully provided an alternative route for photoporation to the highly divergent Gaussian beam. A comparison was given between the two beam approaches for photoporation. The “non-diffracting” character of the BB resulted in the first successful attempt towards automating optical transfection. This was achieved by using an axicon and a spatial light modulator (SLM) to provide phase modulation on the annular spatial spectrum field of the BB. This approach provided control over the lateral and axial position of the beam with respect to the cell membrane, allowing for point and click photoporation. Successful photoporation of CHO cells was also demonstrated using for the first time an axicon tipped optical fibre. The applicability prospects of this method are significant, ranging from potential endoscopic embodiments of the technique to advanced studies of tissue properties in vitro and in vivo.